The Eya family of proteins is essential co-activators of the Six1 transcription factor. Six1 is a developmental gene that is abnormally re-expressed in a large percentage of breast cancers. This over-expression plays a causal role in the initiation and metastatic development of breast cancers. The Eya family of proteins was also found to contain a unique HAD phosphatase domain with protein Tyr phosphatase activity which can potentially play a role in Six1-mediated breast tumorigenesis. Recently, Eya was found to dephosphorylate the histone variant H2AX and direct cells to the DNA repair instead of apoptosis pathway in the event of DNA damage. We have setup a fluorescence-based HTS assay targeting the phosphatase activity of Eya. We propose to perform a large scale high throughput screening using the NIH MLPCN compounds to identify inhibitors of the Eya phosphatase. We plan to test these inhibitors for their potential as therapeutic tools to inhibit Six1-mediated breast tumorigenesis and to increase the efficiency of radiation and certain chemotherapy. These inhibitors can also be used as chemical probes to study the function of Eya's phosphatase activity and its role in Six1-mediated breast tumorigenesis. PUBLIC HEALTH RELEVANCE: The Eya family of proteins is essential co-activators of the Six1 transcription factor which is known to be critical for the onset and progression of a large percentage of breast tumors. In addition, Eya protein contains phosphatase activity that is essential for directing cells to the repair instead of apoptosis pathway upon DNA damage. This phosphatase may also play a role in Six1-mediated breast tumorigenesis. This project aims at identifying inhibitors of the Eya phosphatase activity, which can potentially serve as therapeutic tools to inhibit Six1-mediated breast tumorigenesis and/or to increase the efficiency of radiation and some chemotherapy.